Citric Acid Cycle

Pratt & Cornely, Ch 14

Overview

• Compartmentalization– Glycolysis: Cytosol– Citric Acid Cycle: mitochondria

Overview

• Glycolysis• Pyruvate dehydrogenase

complex– Commitment of carbon away

from carbohydrates• Citric acid cycle

Pyruvate Dehydrogenase Complex

• Three distinct enzymes—in a massive complex• Five chemical steps• What cofactors needed?

Pyruvate Dehydrogenase (E1)

• TPP cofactor: Decarboxylation of a carboxyketones

• Stabilization of “acyl anion”

• Draw mechanism of decarboxylation

Dihydrolipoamide Acyltransferase (E2)

• Transfer catalyzed by E1

• Serves as an linker to “swing” substrate through subunits

• Mechanism of redox

Step 3: transfer

• Maintenance of high energy bond• Acetyl CoA product is made• Lipoamide still reduced—not catalytically

viable at this point

Dihydrolipoamide dehydrogenase (E3)

• Redox of prosthetic FAD/FADH2

• Still not a regenerated catalyst!

Step 5: NADH produced

• Prosthetic group is restored by NAD+• Step 1 uses proton, step 5 regenerates• Oxidation energy of one carbon atom used to– Produce high energy thioester– Produce NADH

Overall Reaction

Problem 6

• Using pyruvate DH as an example, propose a mechanism for the TPP dependent yeast pyruvate decarboxylate reaction in alcohol fermentation.

Fate of Acetyl CoA

• Storage of energy as fatty acid• ATP production (harvest of high potential

electrons)• Formal reaction:

Citric Acid Cycle

• Cyclic pathway– CO2 production

• Substrate level NTP• NADH stores high energy

electrons– Oxidation of alcohol or

oxidative decarboxylation

• QH2 strores high energy elctrons– Alkane reduction to pi

bond

Overview

Carbon Flow

• Each cycle is net oxidation of acetyl CoA– Not actual loss of carbon

from acetyl CoA

• C-13 incorporation experiments

• 4-carbon compounds act “catalytically” in oxygen consumption– Cyclic pathway!

1. Citrate Synthase

• Highly exothermic—lysis of high energy bond

• Used to drive reaction in presence of small [oxaloacetate]

2. Aconitase

• Citrate is achiral and prochiral• Green represents carbon from acetyl CoA– How can enzyme distinguish prochirality?

Prochirality

• Only one compound produced

X

3. Isocitrate Dehydrogenase

• Oxidative decarboxylation

• Spontaneous in b-ketoacids

• NADH• a-ketoglutarate

is a key intermediate

4. a-Ketogluterate Dehydrogenase Complex

• Analogous to pyruvate dehydrogenase complex

• Second decarboxylation, but this is a-decarboxylation

• High energy bond retained

Problem 28

• A patient with a aKG DH deficiency exibits a small increase in [pyruvate] an a large increase in [lactate] so that the [lactate]/[pyruvate] ratio is many times larger than normal. Explain.

Carbon Review

5. Succinyl CoA Synthetase

• Synthetase means ATP (GTP) involved• High energy bond used to do substrate-

level phosphorylation– Good leaving group to activate Pi– Covalent catalysis– GDP GTP

Notice: symmetricalProduct! We lose track of which carbons are from acetyl CoA!

6. Succinate Dehydrogenase

• Oxidation to form C=C releases less energy• FAD is bound redox reagent• In turn, Q is reduced

CO2

O2C

CO2

O2C

FAD

FADH2

O

O

H3CO

H3CO

CH3

R

OH

OH

H3CO

H3CO

CH3

R

Q

QH2

succinate

fumarate

• Q is membrane bound cofactor—revisit in chapter 15!

7. Fumarase

• Another prochiral molecule—makes L-malate• Hydration reaction sets up another oxidation

8. Malate Dehydrogenase

• Large standard free energy• Driven by low [oxaloacetate]– Coupled back to reaction #1

Carbon Flow

• Practice C-14 labeling problems using this basic chart

ATP Harvest: By Enzyme

Net ATP Harvest from Glucose

• Glycolysis = 5-7 ATP– 3 or 5 ATP from

cytosolic NADH– In humans, cytosolic

NADH transport costs 2 ATP equivalents

• Pyruvate DH = 5 ATP• Citric Acid Cycle = 20

ATP• Total: 30 ATP/glucose

in humans

Regulation

• Flux is generated through three irreversible steps

• NADH inhibits • Citrate: product inhibition• Succinyl CoA is last

irreversible product—feedback inhibition

• Ca+2: hormone mediated signal for need for energy

• ADP: need of energy

Anabolic Roles for CAC

• Intermediates can be used for building– Amino acids

• aKG glutamate

– Gluconeogenesis• Through oxaloacetate• Glucogenic amino acid• Acetyl CoA and ketogenic

precursors cannot be used to make net glucose

– Fatty acids• Require transport of

citrate

Citrate Transport System

• Fatty acid biosynthesis happens in the cytosol

• Acetyl-CoA cannot get across the mitochondrial membrane

• At cost of 2 ATP, acetyl-CoA gets across membrane in citrate form

Anaplerotic Reactions• Replenish CAC

intermediates• “Filling up” reactions

– Enhanced aerobic respiration (increase flux)

– Gluconeogenesis pathway

• Key Reaction: Formation of oxaloacetate by pyruvate carboxylase

• Some amino acids can also serve if in high concentration

Problem 42

• Why is the activation of pyruvate carboxylase by acetyl-CoA a good regulatory strategy?

Pyruvate Stimulates CAC

• Some amino acids boost flux by making more CAC intermediates

• Transamination • High [pyruvate]

at beginning of glycolysis boosts flux through CAC

Glyoxylate Pathway

• Makes acetyl-CoA into oxaloacetate in non-cycle

• Allows plants (seeds) to use stored fat to make net glucose

• At expense of bypassing oxidation reactions (NADH production)

Problem 55• Animals lack a glyoxylate pathway and cannot convert fats to

carbohydrates. If an animal is fed a fatty acid with all its carbons replaced by C-14, some of the labeled carbons later appear in glucose. How is this possible?